title: Triterpenoid pristimerin induced HepG2 cells apoptosis through ROS-mediated mitochondrial dysfunction.
abstract: OBJECTIVE
  To investigate the anticancer properties implicated in a natural triterpenoid (pristimerin)-induced apoptosis and inhibited proliferation in human hepatocellular carcinoma (HCC) HepG2 cell line.
  
  
  METHODS
  The cytotoxic activity of pristimerin in HepG2 cells was determined by MTT assay. Apoptotic morphology was observed by fluorescence microscope with Hoechst 33258 staining and percent apoptosis was measured by annexin V/PI double staining. DiOC6 for mitochondrial potential (ΔΨm) and DCFH-DA for reactive oxygen species (ROS) were determined by flow cytometry. Changes of apoptotic- related proteins were analysed by Western blot.
  
  
  RESULTS
  Pristimerin exerted a potent cytotoxic effect on HepG2 cells. After HepG2 cells were treated with pristimerin, typical apoptotic bodies, increasing the proportion of apoptotic annexin V-positive cells and activation of caspase-3 were detected in a dose-dependent manner. It was intriguing that pristimerin increased the generation of ROS with a collapse of the mitochondrial membrane potential in the cells. In addition, there was significant change in other mitochondrial membrane proteins triggered by pristimerin, such as Bcl-2 and Bax. Pristimerin also effectively induced subsequent release of cytochrome C from mitochondria into the cytosol, downregulated EGFR protein expression and inhibited downstream signaling pathways in HepG2 cells. Pretreatment with N-acetylcysteine (NAC) blocked ROS generation and resulted in loss of mitochondrial membrane potential, release of cytochrome C and apoptosis induced by pristimerin.
  
  
  CONCLUSIONS
  These data indicate that ROS play an essential role in the induction of apoptosis by pristimerin in HepG2 cells.
authors.affiliation: Wuhai Municipal People's Hospital, Wuhai, Inner Mongolia, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Yan
authors.forename: Cun-Gen
authors.initials: CG
authors.lastname: Ma
authors.forename: Xia
authors.initials: X
authors.lastname: Wang
authors.forename: Chen
authors.initials: C
authors.lastname: Wang
authors.forename: Jia-Li
authors.initials: JL
authors.lastname: Zhao
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000378:metabolism; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D053978:Pentacyclic Triterpenes; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014315:Triterpenes / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D000975:Antioxidants; C494826:BAX protein, human; D053978:Pentacyclic Triterpenes; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D014315:Triterpenes; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; C512478:EGFR protein, human; D066246:ErbB Receptors; C050414:celastrol